Hemoglobin (Hb), the oxygen-carrying protein in erythrocytes transports oxygen from respiratory organs such as respiratory tracts and lungs and releases oxygen to organs and peripheral tissues of a human body such that the organs and the peripheral tissues can be supplied with sufficient oxygen in order to maintain normal physiological functions.
Hemoglobin of human adults is a tetramer α2β2 consisting of four subunits, α1, α2, β1 and β2, wherein each subunit relies on intermolecular interactions such as intra-subunit hydrogen bonds to sustain its secondary and tertiary structures. Additionally, the inter-subunit hydrogen bonds formed among the aforementioned four subunits allow the quaternary structure of hemoglobin to be formed.
Hemoglobin can reside in two different quaternary configurations, including the relaxed form (R form) having high oxygen affinity and the tense form (T form) having low oxygen affinity. When hemoglobin is travelled to lungs through the blood circulation, hemoglobin becomes bound with oxygen and resides in the R quaternary configuration of high oxygen affinity. The oxygenated hemoglobin is then transported to organs and peripheral tissues through blood circulation and releases oxygen to organs and peripheral tissues and transforms into the T quaternary configuration of low oxygen affinity. The allostery of hemoglobin is also influenced by several allosteric factors, such as the pH value, the carbon dioxide concentration and the 2,3-BPG concentration in erythrocytes. Only when the ratio and spatial arrangement of each subunit of the hemoglobin are correct can the hemoglobin perform its normal biological function of transporting oxygen accurately in the body. Once the subunit ratio of hemoglobin is changed, or the tertiary/quaternary structures are altered, causing defect oxygen transport of hemoglobin and related hemoglobinopathies and blood diseases.
2,3-bisphosphorglycerate (2,3-BPG, or 2,3-diphosphoglycerate, 2,3-DPG; hereinafter “2,3-BPG”) is the endogenous allosteric effector of hemoglobin and the most important chemical species in an erythrocyte of a human body besides the oxygen-carrying entity, hemoglobin. 2,3-BPG delicately regulates the configuration of hemoglobin by interacting with the β1 and β2 subunits of hemoglobin to stabilize hemoglobin in the low oxygen affinity T form to reduce the oxygen affinity of hemoglobin, thereby facilitating hemoglobin to effectively release oxygen to body organs and tissue cells.
Thalassemia is a series of recessive genetic blood disorders and a congenital disease caused by defects in globin chains, and such patients have lower contents and dysfunctional hemoglobin in the body. Thalassemia is also known as Mediterranean anemia. Although there are various types of thalassemia, the most important two types are α-thalassemia and β-thalassemia.
“β-thalassemia” is caused by insufficient synthesis of β-globin chains. Patients suffering from this type of blood disease do not develop significant symptoms during the embryonic development and newborn stages because hemoglobin in embryos or newborn babies is primarily “fetal hemoglobin (HbF).” When the newborns are grown to three to six months old, fetal hemoglobin (HbF) is gradually replaced by adult hemoglobin (HbA), and insufficient production of β-globin chains leads to insufficient production of normal hemoglobin HbA and the patients begin to develop symptoms of anemia such as pale, loss of appetite and loss of vitality. Because children suffering from this type of β-thalassemia cannot produce sufficient β-globin chains in their bodies, those who develop severe symptoms either die early or require bone marrow transplants to sustain their lives, and those who develop mild or moderate symptoms require lifelong periodical blood transfusions and long-term drug therapies to maintain their normal lives.
Sickle cell disease (SCD) is a general term for a group of genetic diseases caused by sickle hemoglobin (Hb S). In many forms of the disease, the shape of red blood cells changes because of the polymerization of abnormal sickle hemoglobin, impairing their ability to carry oxygen. This polymerization process of Hb S causes damages to the cell membrane of erythrocytes, blocks blood vessels, deprives downstream tissues of oxygen and leads to ischemia and infarction. Sickle cell disease is a chronic disease. While patients of Sickle cell disease can generally lead a normal life, they may from time to time suffer from periodical pain. The average life expectancy of these patients is shortened to about forty years. The Sickle cell disease is common in areas where malaria was once epidemic or is still epidemic. It is particularly popular in the population of sub-Saharan Africa, Caribbean, India, the Middle East and Mediterranean, especially Greece and Italy. Nevertheless, the Sickle cell disease may occur on anyone in the world, regardless of races.
Although β-thalassemia and sickle-cell anemia are common blood diseases, there remains no effective treatments up to date. Patients suffering from these blood diseases often rely on blood transfusions, and then take iron chelators to reduce the side effects caused by excessive blood transfusions, such as iron poisoning. On the other hand, the long-term blood transfusion has its drawbacks, including possible shortage of blood sources, limited shelf life of donated blood and even fatal infectious diseases caused by occasional careless blood transfusions.
Therefore, blood substitutes, which can substitute the biological function of normal hemoglobin in transporting and releasing oxygen, have been considered as a new and emerging treatment strategy for above-mentioned blood diseases in recent years. For example, it has been suggested that β-thalassemia and sickle cell anemia can be treated by reactivating or increasing the amount of fetal hemoglobin (HbF) in the body (Blood, 111, 421-429 (2008), Blood, 118, 19-27 (2011)).
At present, there exists three types of blood substitutes: perfluorocarbons (PFCs) cross-linked blood substitutes, hemoglobin-based blood substitutes and recombinant stem cell blood substitutes. Because the structures of the hemoglobin-based blood substitutes are more similar to the structure of the normal hemoglobin than the other two types, the hemoglobin-based blood substitutes are currently the most widely discussed type of blood substitutes.
In the categoty of the hemoglobin-based blood substitutes, hemoglobin variants (Hb variants) and recombinant hemoglobin (recombinant Hb, rHb) are usually suggested to be used as the blood substitutes to replace the function of normal hemoglobin in transporting and releasing oxygen, wherein the fetal hemoglobin (HbF) is the most widely discussed type of hemoglobin-based blood substitute. Just like a normal oxygen delivery system which includes the normal hemoglobin and the allosteric modulators such as 2,3-BPG to aid hemoglobin to properly release oxygen, a sound biomimetic oxygen delivery system must have a main oxygen carrying entity and an effective allosteric modulator which supports and modulates the oxygen carrying entity to release oxygen.
Although the structures of the Hb variants or the recombinant Hb to be used as the blood substitutes are similar to the structure of the normal adult hemoglobin, subtle but crucial difference exists, making them unable to entirely substitute the oxygen carrying and releasing functions of the normal adult hemoglobin. For example, the structure of fetal hemoglobin (HbF) is α2γ2. Though βHis143 is an important active site on the β subunits of normal hemoglobin (HbA) to interact with 2,3-BPG, however, βHis143 is substituted by γSer143 on γ subunits of fetal hemoglobin (HbF), resulting into reduced strength of 2,3-BPG to interact with the fetal hemoglobin (HbF) thus cannot interact with 2,3-BPG normally. Consequently, 2,3-BPG cannot serve satisfactorily as the allosteric modulator for fetal hemoglobin (HbF). This is so, because the oxygen affinity of the fetal hemoglobin (HbF) is too high, which in turn reduces the oxygen release efficiency as compared to that of adult hemoglobin (as shown in FIG. 1). Because the structural modifications for hemoglobin variants and recombinant Hb usually occur at β (non-α) subunits of Hb, 2,3-BPG, which functions by interacting with the two β subunits of hemoglobin thereby cannot modulate the oxygen affinity of these blood substitutes as effectively as that on the normal hemoglobin. Consequently, these Hb-based blood substitutes cannot fully replace the function of normal hemoglobin.